Total RNA from liver and small intestine biopsies was extracted using the RNeasy Plus Mini Kit (Qiagen GmbH, Hilden, DE) according to the indications provided by the company. A small aliquot of total RNA obtained (3 μl) was subjected to qualitative and quantitative control by using the microdrop (Thermo Fischer Scientific, Waltham, MA). The qualitative and quantitative assessment of the individual samples was determined using a dedicated software. The total RNA was reverse transcribed into cDNA by using iScript RT (Bio-Rad Laboratories, Hercules, CA)82 (link). SYBR Green gene expression assays were performed according to the manufacturer’s instruction using the iQ™ SYBR® Green Supermix (Bio-Rad Laboratories, Hercules, CA) and the CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA).
Primer sequences are reported in Supplementary TableS6 .
mRNA expression levels were normalized to β2-microglobulin and quantification of relative gene expression, presented as percentage of the relevant baseline, was calculated using the 2-∆CT (comparative threshold) method.
Primer sequences are reported in Supplementary Table
mRNA expression levels were normalized to β2-microglobulin and quantification of relative gene expression, presented as percentage of the relevant baseline, was calculated using the 2-∆CT (comparative threshold) method.
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